Abstract Book 2010 - CIMT Annual Meeting

029 Brix | Immune monitoring
Culturing PBMC in the presence of antigen results in a measurable
expansion of memory T cells
Liselotte Brix, Tina Jakobsen, and Henrik Pedersen
Immudex, Copenhagen, Denmark
70
Sensitive and reliable monitoring of cellular immune
responses is becoming increasingly important in
cancer vaccine and immunotherapeutic development.
Flow analysis using conventional fluorescent
MHC multimers like Tetramers and Pentamers has
made a great impact in this field enabling visualization,
enumeration and phenotypic characterization
of antigen-specific T-cells. However, shortcomings
such as difficulties in detecting low-affinity interactions,
e.g. tumor-specific T-cell responses, low
reproducibility and reagent stability are still prominent
problems.
MHC Dextramer reagents are a new generation of
fluorescent MHC multimers that solve some of those
problems. Thus, we have used MHC Dextramers to
detect low-affinity T cells (i.e. T cells carrying TCR
with low affinity for the MHC-peptide complex).
In contrast to Tetramers, the Dextramers allowed
enumeration of T cells carrying TCRs with a Kd of
> 250 µM. To our knowledge this is the first time
that antigen-specific T cells with such low-affinity
TCR have been detected with a fluorescent MHC
multimer reagent.
When staining T cells of higher affinity-TCR’s (5
µM – 100 µM), Dextramers had much higher resolution
than the corresponding Tetramers. Dextramers
thus provide a more reliable means for identification
of true positive antigen-specific T cells.
The increased sensitivity and resolution of the
Dextramers probably reflects the higher number of
MHC-peptide complexes and fluorochromes, since
the high number of MHC-peptide complexes increases
their avidity for the specific T-cells, and the
higher number of fluorochromes enhances staining
intensity. As a result, Dextramers have higher sensitivity,
higher staining intensity and higher signalto-noise
ratio.
The dextran backbone of the MHC Dextramer
stabilizes the attached proteins. When analyzing
the stability of the MHC Dextramers, a given
blood sample could be stained reproducibly well
using the same batch of a MHC Dextramer for
more than 4 month. Likewise, different batches
of MHC Dextramers provided similar frequencies
of antigen-specific T cells with comparable staining
intensities when staining the same sample.
We also investigated the impact of freezing and
thawing MHC Dextramers. Surprisingly, the MHC
Dextramer reagent could be subjected to repeated
freeze-thaw cycles without affecting the staining
intensity, resolution or the number of positive cells
detected.
In summary, MHC Dextramers are sensitive, highly
stable reagents that are suitable to minimize assayto-assay
variation, and that are particularly useful
reagents in the immune monitoring of cancer immunotherapy.